The establishment of the segmentation pattern in Drosophila embryos will be investigated. The powerful classical and molecular genetic techniques available in this system make this an excellent model for investigating the mechanism of body pattern formation in a multicellular organism. These studies will provide insight into fundamental aspects of early development and could impact on our understanding of the nature of birth defects. The proposal focuses on defining the role of the runt gene. This is the one of the genes required early in Drosophila development for normal segmentation. These segmentation genes have been grouped into three classes. Genetic experiments reveal a hierarchy of regulatory interactions between these classes with the flow of information going from the "gap" genes through the "pair-rule" genes and finally to the segment- polarity" genes. Runt is a pair-rule gene and has been termed a primary pair-rule gene because of its key role in regulating all of the other pair-rule genes. As is the case for the others, runt shows a pair-rule pattern of expression at the blastoderm stage, because of runt's primary role it is important to understand how this pattern of expression is generated. We will identify cis-regulatory elements of the runt gene that are critical for its proper regulation. This will be accomplished using germ-line transformation to assay DNA elements for their regulatory effects. Trans-acting factors involved in regulating runt will be identified through genetic and biochemical experiments and the mechanism of regulation will be investigated in vitro. The other major aspect of this proposal addresses the function of the runt protein itself and how it acts to regulate other genes. The importance of this issue is emphasized by the observation that runt is unlike all of the other early-acting regulatory segmentation genes and appears to encode a secreted glycoprotein instead of a nuclear transcription factor. We will determine the subcellular localization of the runt protein and investigate the role post-translational modifications play in regulating its activity. An in vitro assay for the function of the runt protein will be used to investigate its mechanism of action. Portions of the protein important for function will be identified by sequencing a collection of mutant alleles of runt and also by sequencing runt homologs isolated from other species. Finally, both biochemical and genetic methods will be used to identify factors that interact with the runt protein and investigate their roles in mediating runt's regulatory activity. This multidisciplinary approach should provide important insight into the role of runt in segmentation and will contribute significantly to our understanding of this basic biological process.